This invention relates to the in vitro cultivation of bacilli and, more particularly, to a novel culture medium capable of being employed for the in vitro cultivation of leprosy mycobacteria.
The causative organism of leprosy is an acid-fast rod, Mycobacterium leprae, which was first described in 1874 by the Norwegian scientist Armauer Hansen. In the more than a century that has elapsed since Hansen's discovery, many investigators have attempted to grow M. leprae in the laboratory for use in leprosy research in testing the effectiveness of proposed new drugs and treatments for leprosy. Although it has been found that rather limited proliferation of M. leprae, generally requiring well over a year for any significant growth, will take place on the footpads of mice and inside armadilloes, the previous efforts to cultivate M. leprae in vitro have been so unrewarding as to have lead to the designation of this pathogen by many investigators as a mycobacterium which is uncultivable in culture media, probably very fastidious in its growth requirements and quite probably an obligate intracellular parasite. Moreover, the pattern of lesion distribution in leprosy patients has also led to a strongly held hypothesis that the growth of M. leprae is low temperature dependent and generally will not occur to any significant degree at physiological temperatures around 37.degree. to 37.5.degree.C.
Recent histochemical studies carried out by the present inventors have led to some novel concepts regarding growth of M. leprae, based on findings that concentrations of M. leprae in the human host are associated with the presence of acid mucopolysaccharides of the host. Mouse inoculation of M. leprae demonstrated that hyaluronic acid applied to the inoculum and inoculation site will promote growth of M. leprae in the mouse abdominal wall and peritoneum, neither of which areas have the postulated required low temperature and neither of which support M. leprae growth in immunologically intact mice. Extracellular bacilli were abundant and after 1 year of such treatment nerve invasion by bacilli was noted. It was also found that a 0.1% saline solution of hyaluronic acid will support viable bacilli indefinitely in the refrigerator or 37.degree.C incubator but with only minimal proliferation. Based on these findings that hyaluronic acid is a suitable basic growth promoter for M. leprae in mice, and presumably is an adequate energy source for M. leprae, it was postulated by the present inventors, contrary to the generally held scientific belief established over the last 100 years, that it might be possible to develop a culture medium, enriched with hyaluronic acid, which would be suitable for the in vitro cultivation of M. leprae.